It has been recently reported that Akt/mTOR signaling pathway is a crucial regulator of skeletal muscle hypertrophy. We have reported that the heat stress accelerates the recovery from skeletal muscle atrophy induced by hindlimb unweighting. Furthermore, we have showed heat stress-induced muscular hypertrophy in both the cultured skeletal muscle cells and the rat skeletal muscles. However, the mechanism responsible for the heat stress-induced muscular hypertrophy remains unclear. In this study, we investigated the role of the Akt signaling pathway in heat stress-induced muscle hypertrophy. Male Wistar strain rats (8-week-old) were mainly divided into two groups; control (Con) and heat stress (Heat). Heat groups were immersed into hot water (42°C) for 15 (H15), 30 (H30) and 60 min (H60). After 1 and 7 days, the plantaris muscles were dissected. Muscle weight-to-body weight ratio of H30 after 7 days was significantly higher than that of Con. The role of Akt signalling pathway in muscular hypertrophy induced by heat stress was examined. [Jpn J Physiol 54 Suppl:S119 (2004)]

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Macromolecules, such as Dextran T500 (Mw 500k), have been considered to compress skinned fibers of skeletal muscle osmotically because they are too large to penetrate into myofilament lattice. However, we found that polyethylene glycols (PEG) of small molecular weight (Mw 3350 and 200) efficiently compress skinned fibers. From the size of the molecule, they are small enough to penetrate into the myofilament lattice. Since PEG was reported to affect interaction between proteins, we pursued the effects of relevant polyols on the volume of skinned fibers at rest. Fiber volume was measured as optical cross-sectional area of isolated skinned fibers from bullfrog sartorius muscle. Any polyols in the ethylene glycol (EG) series (EG, diEG, triEG, tetraEG, PEG200, PEG900, PEG3350, and cyclodextrins) compressed the fibers. Most efficient was PEG3350 (Mw 3350), which compressed the fiber volume to 25% of the original at 30% (w/w). Interestingly, even on mole fraction basis, EG (H-[CHOH]₂-H) was as efficient as threitol (H-[CHOH]₄-H), but more efficient than glycerol (H-[CHOH]₃-H). Cyclodextrins (Mw 972-1297), which has characteristic molecular structure, were roughly as efficient as PEG200. PEG suppressed isometric force development of the fiber, while cyclodextrins significantly increased the isometric force as well as the rate of force rise on activation with Ca²⁺. These results indicated that the effects of polyols on skinned fibers were not a simple osmotic effect but reflect subtle structure of the molecules. The effect of macromolecules on muscle should be carefully reconsidered in this light. [Jpn J Physiol 54 Suppl:S119 (2004)]

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We gravimetrically observed evaporation of myowater components from skinned skeletal muscle. Fiber bundles of around 30-130 mg dissected from sartorius muscle of bullfrogs were skinned chemically with 0.5% Triton X-100. The specimen was equilibrated with the controlled humidity at room temperature. The humidity was gradually decreased from 95 to 5%RH. At 85-90%RH, about 60% of myowater was evaporated from the muscle. The second peak was found at 75%RH, where 5-10% was evaporated. Continuous gradual evaporation was followed with intermittent peaks found at 55 and 48%RH. At each of the humidity ranges of 74-55%RH and 55-48%RH, 5-10% of myowater was evaporated. There remained 15-25% of myowater at the lowest humidity of 5-15%. This result indicated that myowater is able to reserve at least up to 3kT of free energy in its state. Parallel observation of spin-spin relaxation of ¹H-NMR signals from water protons (spectrometer: varian, Gemini 2000-300BB) indicated that myowater evaporates in the order of its relaxation time constant (T₂). From the order of evaporation and the relative volume of water components characterized by characteristic T₂ values, the component of T₂ around 0.15 s corresponds with water evaporated at 85-90% RH. The component of 10^−1.5<T₂<10⁻¹ s is considered to evaporate at 74-55%RH and the more rapidly relaxing component would evaporate at 55-48%RH. This assignment confirmed that myowater components resolved by spin-spin relaxation possess characteristics substantially distinct from each other. [Jpn J Physiol 54 Suppl:S120 (2004)]

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The Ca²⁺-independent contraction of vascular smooth muscles (VSMs) mediated by Rho-kinase plays a central role in the pathogenesis of abnormal contraction of VSMs such as vasospasm. Previously, we identified SPC (sphingosylphosphorylcholine) as a novel signaling molecule which causes Ca²⁺-independent VSM contraction through the activation of Rho-kinase, without the increase in intracellular Ca2+ concentration ([Ca²⁺]_i). Furthermore, we identified EPA (eicosapentaenoic acid) as a specific inhibitor of the Ca²⁺-independent contraction. Here we examined the effects of EPA on the [Ca²⁺]_i and the tension of VSMs, both of which were measured simultaneously by loading VSM strip with fura-2. SPC induced VSM contraction without changing [Ca²⁺]_i, whereas high K⁺-depolarization and U46619 induced VSM contraction with the increase in [Ca²⁺]_i. When the contraction by those stimuli reached the maximum and steady state, EPA was applied. EPA had an inhibitory effect on the contraction induced by SPC and U46619, but did not inhibit the contraction induced by high K⁺-depolarization. Interestingly, EPA had an inhibitory effect on the [Ca²⁺]_i increase induced by U46619, but not on the [Ca²⁺]_i increase induced by high K⁺-depolarization. In this study, we also analyzed the steric effects of EPA on these vascular actions. [Jpn J Physiol 54 Suppl:S120 (2004)]

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We have been developing a sarcomere model to account for the physiological Ca release from the junctional sarcoplasmic reticulum (JSR) in the cardiac myocyte based on the Ca-induced Ca-release mechanism of the JSR without any inactivation of the Ca release channel. In the present study, we constructed a cylindrical three-dimensional sarcomere model with the transverse (T)-tublule, the diad, the JSR and the tubular (T)- SR and examined the following problems by simulating the physiological JSR Ca release and a staircase: (1) Where is the Na/Ca exchange localized in the T-tubule? (2) Where does Ca enter into the cytoplasm for replenishing the JSR in diastole? (3) Is Ca uptake activity of the JSR necessary? (4) How fast does Ca move from the T- to the JSR? Simulation was made successfully by assuming (1)(2) the localization of the Na/Ca exchange and the diastolic Ca influx at the diad, (3) a low rate of Ca uptake by the JSR to inhibit the JSR Ca release after the termination of the action potential-dependent Ca influx by lowering the [Ca] in the diadic cleft together with a high rate of Ca uptake by the TSR and (4) slow movement of Ca from the T- to the JSR. We present the [Ca] distribution in the sarcomere at some selected times after the JSR Ca release and the change of the [Ca] with time at some selected points in the sarcomere together with the change of the average [Ca] in the cytoplasm. [Jpn J Physiol 54 Suppl:S120 (2004)]

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Our AFM studies revealed that the transverse stiffness and its distribution are different for cardiac and skeletal myofibrils and it was differently affected by proteolytic treatments for the two myofibrils. To give a molecular basis for these findings, in the present study, we investigated how differently molecular components of cardiac and skeletal myofibrils were digested by proteolytic treatments.
Single myofibrils were prepared by homogenizing glycerinated muscle fibers of the left ventricle of rat heart and the rabbit psoas muscle. Proteolytic digestions of myofibrils were made by calpain and trypsin. SDS-PAGE was performed on 2.5%~12% gradient gels.
By the calpain treatment, α-actinin in cardiac and skeletal myofibrils was degraded although its rate was slow for cardiac myofibrils and very fast for skeletal myofibrils. Connectins in both myofibrils and nebulin in skeletal myofibrils were also degraded while other muscle proteins were almost not degraded. By the trypsin treatment, connectin in both myofibrils and nebulin in skeletal myofibrils were degraded although their degradation products were different for cardiac and skeletal myofibrils. And α-actinin and other muscle proteins were almost not degraded. These results suggested that the difference in the mechanical characteristics between cardiac and skeletal myofibrils is based on the difference in the molecular structures of the two myofibrils. [Jpn J Physiol 54 Suppl:S120 (2004)]

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Calcineurin, a calcium-regulated serine/threonine phosphatase, has been reported to be one of the signaling molecules in cardiac hypertrophy, yet it is currently under investigation whether its pathway is implicated in the phenotypic regulation of cardiac muscle. To elucidate this question, we compared the expression pattern of myosin heavy chain (MHC) isoforms (alpha- and beta-MHCs) in right (RV) and left (LV) ventricules obtained from control and cyclosporin A (CsA, a calcineurin inhibitor)-treated rats. Rats, aged 8 weeks, were injected i.p. with normal saline (control group) or 10 mg/kg CsA (CsA-treated group) daily for 2 weeks. Alpha- and beta-MHCs were separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their relative proportions were densitometrically quantified. The relative proportions of alpha- and beta-MHCs (mean±SD, n=4) were 70.7±3.6％ and 29.3±3.6％ in RV, 72.0±3.9％ and 28.0±3.9％ in LV of control group and 80.0±1.4％ and 20.0±1.4％ in RV, 79.5±2.4％ and 20.5±2.4％ in LV of CsA-treated group, indicating a significant (p<0.05) increase in the relative proportion of alpha-MHC at the expense of that of beta-MHC in both right and left ventricules in association with the CsA treatment. These results suggest that calcineurin pathway is implicated in a conversion of cardiac muscle towards more beta-MHC-rich phenotype. [Jpn J Physiol 54 Suppl:S121 (2004)]

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Molecular dynamics study on dynamical structures of mutant cardiac troponin T, which have been reported to be involved in serious cardiac diseases, were carried out. Calculations were done on troponin T fragment (residue 204 to 230) using Software Amber ver.7 based on a crystal structure of human troponin CIT complex in Protein Data Bank (1J1E). Iteration was done in TIP3 water sphere (25 angstrom of diameter) with 0.002 ps time step in non periodic condition at constant temperature (310 K). A mutant troponinT which lacks K210 (ΔK210) is one of the causes of familial dilated cardiomyopathy (DCM) and skinned fiber in which this mutation was introduced has been reported to have reduced calcium sensitivity (Morimoto et al. 2002). According to the analysis by chou fasman protein secondary structure prediction, we made a ΔK210 mutant model of troponin T fragment to keep forming alfa helix by shift and the rotation (1/3.6 turn) of the residues in N terminal side connected to the deleted one. We found that the dynamical structure of this mutant model showed two characteristics compared with that of wild type, that is; 1) an alfa helix formed by residue 204 to 223 shrank by about quarter turn as a direct result of deletion, and 2) acidic side chain of G204 and basic side chain of K208 exhibited strong electrostatic interaction. These changes observed in this model may modify the mobility and orientation of N terminus of troponin T (TnT1) and C terminus of troponin T (TnT2), which are thought to be essential for regulation of muscle contraction. [Jpn J Physiol 54 Suppl:S121 (2004)]

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It is well known that the muscle produces more force at a longer sarcomere length (SL). By x-ray diffraction study (at BL45XU in SPring 8), the equatorial reflection ratio of I(1,0) and I(1,1) was well related to the peak tension in rat papillary muscle, showing that a larger tension accompanies a smaller ratio. Thus, the tension, SL, and ratio are all related to each other: at a longer SL, the twitch tension is larger and the intensity ratio smaller. It is predictable that the peak tension and the I(1,0)/I(1,1) intensity ratio in systole are also related to each other since both are related to the SL. However, these two parameters are more strongly correlated. In a small range of SL, the peak tension is larger when the intensity ratio is smaller, showing that this relation holds regardless of the SL. When the same data points were classified into three groups by the peak tension, no significant correlation was found between the SL and the I(1,0)/I(1,1) intensity ratio within each group. Similarly, no correlation was found between the twitch tension and the SL when the data were classified into three groups by the I(1,0)/I(1,1) intensity ratio. Thus, the correlation between the intensity ratio and the tension, is not mediated by the SL, but holds even when the tension varies depending on the specimen at the same SL. This is consistent with the interpretation that the intensity ratio in systole is related to the number of cross-bridges formed in contraction. [Jpn J Physiol 54 Suppl:S121 (2004)]

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The intrinsic mechanical properties of intact cells are basically important themselves and also important to explain those of muscle bundles, since the muscle bundles are comprised of infinitely many cells. The mechanical properties of single cardiac cells enzymatically isolated from rat ventricles were studied. Both ends of single cells were firmly and stably glued to the glass-fiber needles (diameter of 30 μm) coated with poly-L-lysine to measure the length change or the force by the handmade electrostatic force-length transducer. The absolute forces were measured in resting state, which is considered to express the parallel elastic element. The resting force seemed to increase with the cell length. The forces were also measured during twitch-contractions. The measured active forces were typically more than 1 μN. The shortening velocity, which is considered to express the contractile element, was measured by various methods, namely, the quick release method (QR), the afterload contraction method (AL) and the ramp-load release method (RAMP) applied the linear load decrement from the time of peak tension developed. The maximal shortening velocities (Vmax) were about 2.6 cell length/sec (CL/s) by QR, 0.9 CL/s by AL. The ramp rate dependent Vmax was 0.5 CL/s by RAMP with the ramp rate of -10 μN/sec. The length change was related rather linearly to the tension change with the decay rate of about 22 μm/μN, which is considered to express the series elastic element. Finally, the main three mechanical elements of single cardiac cells were obtained. [Jpn J Physiol 54 Suppl:S121 (2004)]

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To investigate the contractile properties of young (4-5 weeks) mouse cardiac ventricular cells, we isolated ventricular cells from 4-5 weeks mice. Ventricular cells were transffered to a non-fluorescent glass chamber, mounted on the stage of an inverted microscope (Eclipse TE2000, Nikon, Japan). Cell length was captured through an objective lens with either 20x or 40x magnification (Fluor 20x, SFluor 40x, Nikon, Japan). Cell shortening was detected with a MyoCam CCD camera at a speed of 60 Hz. The video-monitored data were digitized through a DSI 1200 computer interface (IonOptix, USA) and were analyzed by a video-edge-detection system (IonOptix, USA). Electrical field stimulation (15 V, 10 ms) was applied through a silver electrode placed near the cell. When extracellular calcium concentration ([Ca]o)was 0.9 mM, myocytes showed the negative staircase of cell shortening at a stimulation frequency of 0.2, 0.5, and 1 Hz. At a stimulation frequency of 0.1 Hz or less, the degree of cell shortening was constant. We then investigated the effecct of [Ca]o on the negative staircase of cell shortening. At [Ca]o of less than 1.8 mM, the cells showed the negative staircase. However, at [Ca]o of greater than 2.5 mM, the negative staircase disappeared. These results suggest that stimulation frequency and [Ca]o are to be considered to evaluate contraction or calcium dynamics in genetically engineered mouse cardiac myocytes. [Jpn J Physiol 54 Suppl:S122 (2004)]

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Purpose: We have investigated structural alterations of the capillary network and vascular apoptosis in the soleus muscle of rats after 2wks of hindlimb unloading. Methods: Ten male Wistar rats (aged 10 wks old) were used. They were randomly divided into a control group (n=5, CONT) and a hindlimb unloading group (n=5, HS). Three-D architecture of microvasculature of the soleus and the apoptosis of vascular endothelial cell were morphometrically examined using confocal laser scanning microscope. Results: The cross-sectional area of muscle fiber and muscle mass were significantly decreased in HS. The number of longitudinal capillaries paralleling muscle fiber did not change with atrophy in the soleus muscle. However, the mean capillary density, the number of anastomotic capillaries and capillary luminal diameter were significantly smaller in HS than in age-matched CONT. In addition, the tortuosity of capillaries was significantly decreased and TUNEL positive nuclei were observed in atrophied muscle. Concluding Remarks: These results indicate that muscle atrophy by unloading the hindlimb generates structural alterations of capillary network, and apoptosis appears to occur in vascular endothelial cell of longitudinal and/or tortuous capillaries. [Jpn J Physiol 54 Suppl:S122 (2004)]

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The aim of the present study was to characterize the prejunctional modulation of evoked tritium outflow in the guinea-pig vas deferens preincubated with [³H]noradrenaline. Vasa deferentia were stimulated electrically by trains of 50 pulses at 40 Hz with 4 mA. The evoked outflow of tritium was enhanced by yohimbine (1 μM, 213±38%, n=6, p<0.0001) and rauwolscine (1 μM, 201±31%, n=4, p<0.01), the α₂ adrenoceptor antagonists and inhibited by clonidine (1 μM, 63±3%, n=4, p<0.05), the α₂ agonist. The P2 purinoceptor antagonists suramin (300 μM, 94±9%, n=5) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 μM, 100±2%, n=4) did not significantly change the tritium outflow. The tritium outflow was enhanced by BK channel antagonists iberiotoxin (100 nM, 127±9%, n=7, p<0.001) and γcharybdotoxin (100 nM, 145±22%, n=10, p<0.001), and decreased by SK channel antagonist apamin (100 nM, 88±8%, n=6, p<0.01). L-type Ca²⁺ channel agonist, Bay K 8644 (1 μM, n=8) and antagonists, nifedipine (40 μM, n=8), nimodipine (10 μM, n=4) and nicardipine (20 μM, n=4) did not affect on the spontaneous and evoked outflow of tritium. N^w-nitro-L-arginine (100 μM, n=6), an inhibitor of NO synthase, and sodium nitroprusside (100 μM, n=6), a NO donor had no significant effect on the evoked outflow of tritium. These results indicate that α₂ adrenoceptors and BK channels play an important role in a prejunctional modulation of neuronal noradrenaline release in the guinea-pig vas deferens. [Jpn J Physiol 54 Suppl:S122 (2004)]

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Two pathways have been proposed for Mg²⁺ transport across the plasma membrane: 1) Na⁺-Mg²⁺ exchange and 2) Na⁺-independent passive Mg²⁺ pathway in visceral smooth muscle (Nakayama et al. J. Physiol.551.3,843-853,2003). Recently, Nadler et al. (Nature411,590-595,2001) has identified LTRPC7 as a Mg²⁺ permeable channel in the plasma membrane. In this study, using 31P-nuclear magnetic resonance (NMR), we measured the intracellular free-Mg²⁺ concentration ([Mg²⁺]_i) in pig carotid artery smooth muscle. [Mg²⁺]_i was estimated from the chemical shift of the β-ATP peak. Intracellular pH (pH_i) estimated from the chemical shift of P_i was used to correct the [Mg²⁺]_i value. We found that [Mg²⁺]_i was increased by increasing the extracellular Mg²⁺ concentration in the absence of Ca²⁺. This was true when extracellular Na⁺ was substituted with equimolar K⁺ or NMDG(N-methyl-D-glucamine). 2-APB is a known potent blocker for LTRPC7. We also examined whether this drug affects the Na⁺-independent [Mg²⁺]_i rise. [Jpn J Physiol 54 Suppl:S122 (2004)]

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We have reported that externally applied ATP induces non selective cation currents in pregnant rat myometrial cells and suggested the currents contribute to spontaneous depolarization leading to uterine contraction (Pflügers Arch. 447, 457-64, 2004). However, the channel that carries the ATP sensitive non selective cation currents have not been molecularly identified in rat uterus. Thus, expression of P2X receptor in mRNA levels was screened with RT-PCR method using cDNA library from pregnant rat myometrium at day 21. Most abundantly expressed P2X4 (more than 99% sequence homology to that of rat brain) among other subtypes (P2X1 through P2X7) was cloned, and functionally expressed in COS-7 or CHO cells. The expressed channels were activated by externally applied ATP as well as other nucleotides (ATP>GTP>UTP>ADP). The activated channels were non selectively permeable to various monovalent cations (K⁺>Cs⁺>Na⁺>Li⁺) and were blocked by non selective P2 receptor antagonist, suramin and selective P2X blocker, PPADS. These properties resembles to those of ATP sensitive non selective cation currents previously reported in pregnant rat uterus. These results, therefore, indicate that P2X4 receptor is the most probable candidate for carrying the ATP sensitive non selective cation currents in pregnant rat myometrium. [Jpn J Physiol 54 Suppl:S123 (2004)]

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It has been suggested that periodic rise of the intracellular Ca²⁺concentration ([Ca²⁺]_i) is a primary mechanism in c-Kit-immunopositive interstitial cells (=ICC), and consequently generating pacemaker potentials through periodic activation of Ca²⁺-activated Cl⁻ channels. We have recently developed a cultured cell cluster preparation from the small intestine of the mice. On the other hand, many important reports have dealt with spontaneous rhythmicity in stomach smooth muscle. Therefore, in this study, we prepared cell clusters from the stomach smooth muscle tissue of mice.
　In the presence of nifedipine, pacemaker ([Ca²⁺]_i)oscillations were observed limited regions with positive c-Kit-immunoreactivity. In the majority of cell cluster preparations with multiple regions of ([Ca²⁺]_i)oscillations, ([Ca²⁺]_i)oscillated synchronously in the same phase. A small number of cell clusters showed multiple regions of ([Ca²⁺]_i)oscillation synchronised but with a considerable phase shift. Applications of either 2-APB or xestospongin C rather rapidly abolished the spontaneous ([Ca²⁺]_i)oscillation, suggesting involvement of intracellular Ca²⁺release channels, especially inositol 1,4,5-trisphosphate receptor. [Jpn J Physiol 54 Suppl:S123 (2004)]

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To determine the cause of dysfunction of colonic movement in patients with Duchenne muscular dystrophy, we studied the effect of selective inhibitor of e-, i- and n-nitric oxide synthase (NOS) on the migrating motor complexes (MMCs) in proximal and distal segments of colon of muscular dystrophy (mdx) mice and compared the findings with those of the control. There was no difference in the frequency of MMCs between the mdx colon and the control; it was 0.58±0.06 (n=9), 0.26±0.04 cpm (n=5) in the mdx proximal and distal segments of colon respectively and 0.72±0.10 (n=6) and 0.29±0.03 cpm (n=7) in the control. The frequency of MMCs was greater in the proximal than the distal segments in both the mdx colon and the control (p<0.01). 1,3-PBIT (inhibitor of i-NOS at low and e-NOS at high dose) and spermine (n-NOS inhibitor) extinguished the MMCs in both the mdx colons and the control. In some quiescent mdx colons and the control, 1,3-PBIT induced the MMCs at a low dose. L-NAME (NOS inhibitor) induced the MMCs in some quiescent mdx colons, but not in the control. The induced MMCs were extinguished by 1,3-PBIT at a high dose or NOC (NO donor). Spermine never induced the MMCs in either the mdx colon or the control. There was no difference in the effect of these NOS inhibitors between the proximal and the distal segments in both the mdx colon and the control. The result suggests that inhibition of i-NOS is more effective than e- and n-NOS in the generation of the MMCs and that the mdx colon is more susceptible to the inhibition of NOS than the control. [Jpn J Physiol 54 Suppl:S123 (2004)]

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Properties of electrical responses evoked by cholinergic nerve stimulation were investigated in single bundle preparation of circular smooth muscle isolated from the guinea-pig gastric antrum. In the absence of nitrergic components by inhibiting the biosynthesis of nitric oxide with nitroarginine, smooth muscle produced periodical generation of slow potentials. Transmural nerve stimulation (TNS) evoked an apamin-sensitive inhibitory junction potential (IJP) and following atropine-sensitive depolarization response, the latter was either a transient depolarization with enhanced generation of unitary potentials or a slow potential, when TNS was applied at the interval between slow potentials. TNS elicited a generation of slow potentials only when it was applied at a certain period of time after the cessation of slow potentials, i.e., there was a high-threshold period for the generation of slow potentials by TNS. The high-threshold period was about 10s after cessation of slow potentials, and it was increased to about 20s in the presence of chelerythrine and decreased to about 6s by phorbol esters. It is concluded that neuronal cholinergic excitation of gastric muscle involves an activation of PKC, and the high-threshold period for the generation of slow potentials by TNS may be causally related with the low activity of PKC. [Jpn J Physiol 54 Suppl:S123 (2004)]

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Heat shock protein 20 (HSP20) has actin binding capacity and its amino acid sequence of actin binding region (residue 110-121) is highly homogenous to the inhibitory region of skeletal muscle troponin I (residue 104-115; TnIp). Recent studies reported contribution of HSP20 phosphorylation in cGMP-induced relaxation, and force inhibiting effects of a syntetic peptide of HSP20 (residue 110-121; HSP20_p) on tonic vascular skinned smooth muscle (Rembold et al., 2000). To elucidate the role of HSP20_p on the phasic smooth muscle, we studied the effects of HSP20_p on contractile properties of Triton-X-100 skinned muscle preparations from guinea pig taenia caeci. In Ca²⁺ induced contraction, HSP20_p enhanced Ca²⁺ sensitivity for the force, although HSP20_p slightly inhibited the maximal Ca²⁺ induced force. Also HSP20_p enhanced Ca²⁺ induced posphorylation of myosin regulatory light chain (MLC20). On the other hand, HSP20_p inhibited the myosin phosporylation independent contraction by 30 mM Mg²⁺. Our present results suggest that, in phasic smooth muscles, HSP20_p may affect both thick filament linked- and thin filament linked-regulation of contraction. [Jpn J Physiol 54 Suppl:S124 (2004)]

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To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles whilst recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca²⁺]_i; Ca transients) were recorded in fura-PE3 loaded strips. Spontaneous action potentials and contractions were abolished by nifedipine (1 μM). Carbachol (0.1 μM) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 μM) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nM) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 μM) reduced the frequency of action potentials and contractions. Forskolin (0.1 μM), 8-bromoguanosin 3', 5' cyclic monophosphate (0.1 mM) and Y-26763 (10 μM) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. These results confirm that action potentials and associated Ca transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca²⁺ may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase [Jpn J Physiol 54 Suppl:S124 (2004)]

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In isolated circular muscle bundle of the guinea-pig stomach antrum, smooth muscle cells had the resting membrane potential of about -70 mV, and they produced periodical generation of slow potentials (amplitude 25 - 40 mV, duration 5 - 7 s, frequency 0.2 - 5/min). Acetylcholine (ACh), 1 - 100 nM, increased the amplitude and frequency of slow potentials with no significant depolarization of the membrane, and the actions of ACh were abolished by atropine. Chelerythrine, 0.1 - 1 μM, reduced the frequency of slow potentials, with no alteration to the amplitude of slow potentials and the resting membrane potential. The ACh-induced increase in frequency, but not the amplitude, of slow potentials was inhibited by chelerythrine. 2-APB, 3 μM, increased the frequency of slow potentials and decreased the amplitude, with depolarization of the membrane. In the presence of 2-APB, ACh increased the frequency of slow potentials, with no significant increase in the amplitude. A known activator of proteinkinase C, phorbol 12, 13-dibutyrate (PDBu), 1 nM, increased the frequency, but not the amplitude, of slow potentials, with no depolarization of the membrane, and the effects were antagonized by chelerythrine. Thus, the ACh-induced increase in the frequency of slow potentials was mimicked by PDBu. The results suggest that ACh increases the frequency of slow potentials by activating proteinkinase C. The amplitude of slow potentials may be related to the amount of Ca released from internal stores through activation of IP₃ receptors. [Jpn J Physiol 54 Suppl:S124 (2004)]

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To investigate the possible contribution of T-type Ca channels to the generation of spontaneous electrical activity in detrusor smooth muscles of the guinea-pig, the effects of blockers for the channels on action potentials were examined using intracellular recording techniques. Isometric tension recording and intracelluar Ca measurement using furaPE3 loaded preparations were also carried out. Two types of action potentials were generated in detrusor smooth muscles of the guinea-pig bladder. Individual type of action potential is readily blocked by nefedipine(1μM) while bursting action potentials were converted into individual depolarizations. Higher concentrations of nifedipine(10-30μM) were required to block the depolarizations. Ni²⁺(100-300μM) changed individual action potential to bursts without changing either membrane potential or dV/dt of action potentials. Mibefradil(1μM), another T-type Ca channel blocker, also changed the pattern of action potential without affecting on their time courses. Consistently Ni²⁺ increase the amplitude of spontaneous contractions and Ca transients, and reduced their frequency. These result suggested that T-type Ca channel may determine the frequency of spontaneous action potential whereas their amplitude mostly depends on the activity of L-type Ca channel. [Jpn J Physiol 54 Suppl:S124 (2004)]

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A synthetic peptide of the inhibitory region of cardiac troponin I [136 - 147] (troponin I inhibitory peptide; TnIp) suppresses Ca ion induced contraction of smooth muscle preparations skinned with beta-escin via direct interference with actin-myosin interaction (Watanabe et al., BBRC 307, 236-40, 2003). To analyse TnIp action on the cross-bridge cycling in detail, we studied TnIp effects on the relaxation of skinned taenia caeci smooth muscle induced by the Ca ion removal with EGTA. In the presence of TnIp at more than 100 micro molar, the tension did not fall to the resting level completely, but instead reached a sustained force maintaining level. Data analysis of the time course of the relaxation indicated TnIp accelerating transformation of rapid- to slow-cross-bridge cycling. These results suggest that thin-filament-liked regulatory systems in smooth muscle may contribute "latch" cross-bridge formation. [Jpn J Physiol 54 Suppl:S125 (2004)]

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We have recently identified the molecular determinants of cardiac-specific use dependent block (UDB) by a class 1c antiarrhythmic agent pilsicainide, which are located at transmembrane segments of IVS6. In the present study, the significance of the sites for other antiarrhythmic agents was investigated. The conversion of amino acids for pilsicainide-responsible sites in rat heart sodium channels (Nav.1.5) to those of the corresponding sites in rat brain channels (Nav.1.2) in HEK293 cells, reduced extents of UDB in heart channels by flecainide and quinidine similar to those in brain channels. Interestingly, UDB by lidocaine was also reduced by the conversion above only when short duration (10ms) repetitive pulses were applied. The voltage-dependency of UDB by lidocaine was well correlated with that in the channel activation, suggesting that lidocaine recognizes open-state high affinity sites under this condition. Taking into account the fact that pilsicainide, flecainide and quinidine are all well-known open-channel blockers, these results indicate the possibility that previously identified pilsicainide-responsible sites could be the common significant sites for antiarrhythmic agents in isoform-specific open-channel block of cardiac sodium channels. [Jpn J Physiol 54 Suppl:S125 (2004)]

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[Aim] We investigated acute effects of sex hormone on membrane currents of cardiomyocytes, and their intracellular signal transduction mechanism. [Methods] Cardiac membrane currents were recorded with amphotericin B-induced perforated patch from guinea-pg ventricular myocytes. Intracellular signal transduction mechanism was investigated by Western blot analysis and immunoprecipitation. [Results] (1) Both 17β-estradiol (E₂) and 17β-dihydrotestosterone (DHT) enhanced slowly-activating delayed rectifier K⁺ current (I_Ks) in a concentration-dependent manner with an IC₅₀ of 10.3±0.2 nM and 1.1±0.1 nM, respectively. I_Ks enhancement by E₂ and DHT was inhibited by a blocker of estrogen receptor (ER), ICI182780, and androgen receptor (AR), nilutamide, respectively. (2) I_Ks enhancement by E₂ and DHT was inhibited by NOS3 inhibitor, but not by NOS1 inhibitor. NOS3 is activated by either Ca²⁺/calmodulin(CaM)- or Ser/Thr kinase, Akt-dependent manner. I_Ks enhancement by E₂ and DHT was inhibited by Akt inhibitor, but not by CaM inhibitor, W7. (3) E₂ and DHT induced phosphorylation of Akt and NOS3. NOS3 phosphorylation was inhibited by Src inhibitor (PP2), PI3 kinase inhibitor (wortmannin), Akt inhibitor, and hsp90 inhibitor (geldanamycin). Akt phosphorylation was inhibited by PP2 and wortmannin, but not by Akt inhibitor or geldanamycin. [Conclusion] E₂ and DHT enhanced I_Ks by NO produced through non-genomic pathway of E₂ and DHT. [Jpn J Physiol 54 Suppl:S125 (2004)]

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Angiotensin II (Ang II) produces numerous short- and long-term effects on cardiac muscle under various pathophysiological conditions. There is, however, little information concerning the immediate electrophysiological effects of Ang II on the cardiac repolarization process. The present study was designed to investigate the effect of Ang II on the slowly activating component of delayed rectifier K⁺ current (I_Ks) in guinea-pig atrial cells using the whole-cell patch-clamp technique. Bath application of Ang II (>=1 μM) increased the amplitude of I_Ks, evoked by 2-s depolarizing steps applied from a holding potential of -50 mV. The stimulatory effect of Ang II was largely abolished by the presence of the AT1 receptor antagonist valsartan (1 μM) and was mimicked by the specific AT1 agonist Sar-Ang II. These results indicate that I_Ks was enhanced via AT1 receptor. The stimulatory effect of Ang II was significantly reduced in cells loaded with nonhydrolyzable GDP analogue GDPβS (1 mM) or the phospholipase C (PLC) blocker compound 48/80. In addition, potentiation of I_Ks by Ang II was markedly reduced by pretreatment of the cells with the protein kinase C (PKC) inhibitors H-7 (10 μM) and BIS (100 nM) but was not affected by intracellular perfusion of BAPTA (20 mM).Based on these experimental results it is concluded that Ang II potentiates I_Ks by stimulating AT1 receptor couple to G protein-PLC-PKC pathway. [Jpn J Physiol 54 Suppl:S126 (2004)]

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It has been demonstrated that sphingosine–1–phosphate (S1P) elicits many cellular responses by binding to G–protein–coupled endothelial differentiation gene–encoded (Edg) receptors. In the heart, S1P activates the muscarinic K⁺ channel (IK,ACh) in sino–atrial (SA) node cells and thereby evokes negative chronotropic actions. In the present study we investigated the effect of S1P on the slowly activating component of delayed rectifier K⁺ current (I_Ks) in guinea–pig ventricular myocytes using the whole–cell patch–clamp technique. I_Ks was elicited by depolarizing voltage steps given from a holding potential of –50 mV to various levels up to +50 mV and the effect of S1P on I_Ks was assessed by measuring the amplitude of the tail current elicited upon return to the holding potential. External application of S1P in nanomolar to micromolar concentrations reversibly increased the amplitude of I_Ks with mean half–maximal concentration (K_1/2) of 69.7 nM (n = 6). S1P at 1 μM evoked a maximal increase of I_Ks by a factor of 1.68±0.1 (n = 6). Preincubation of ventricular myocytes with pertussis toxin (PTX, 5 μg/mL) largely abolished the stimulatory action of S1P, suggesting that a G protein–coupled plasma membrane receptor for S1P is involved. Thus the present investigation identifies a novel extracellular signalling molecule that positively regulates I_Ks in mammalian ventricular myocytes. [Jpn J Physiol 54 Suppl:S126 (2004)]

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Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects diverse cellular functions such as cell proliferation and differentiation. In recent years it has also been shown that many of these actions are mediated through the G protein-coupled plasma membrane receptors highly selective for LPA. There is, however, little information concerning the acute electrophysiological effects of LPA on ion channel functions in the heart. The present study was designed to examine the effect of LPA on the slow component of delayed rectifier K⁺ current (I_Ks) in guinea-pig atrial myocytes using the whole-cell patch-clamp method. Extracellular application of LPA reversibly increased I_Ks, elicited by 2-s depolarizing voltage pulses to various levels (up to +50 mV) applied from a holding potential of -50 mV. The stimulatory effect was concentration-dependent with mean half-maximal concentration (K_1/2) of 3.9 nM, and the maximal effect (increase by 107.4±15.2%, n = 14) was obtained by 1 μM LPA. The LPA-induced enhancement of I_Ks was markedly attenuated by intracellular loading with nonhydrolyzable GDP analogue GDPβS (2 mM) but was not significantly affected by pretreatment with pertussis toxin (PTX, 5 mg/ml). These results strongly suggest that the stimulatory effect of LPC is mediated through the plasma membrane receptor coupled to a PTX-insensitive G protein. The present observation that I_Ks receives a positive regulation by LPA indicates that LPA can produce acute action on the repolarizing process in cardiac myocardium. [Jpn J Physiol 54 Suppl:S126 (2004)]

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Background Nifekalant is a blocker of cardiac I_kr channels encoded by a HERG gene and is reported to be clinically effective for lethal ventricular tachyarrhythmias. However, nifekalant-binding sites in a HERG channel have not been identified. Thus, we attempted to identify these sites and compare them with those for MK-499, a methanesulfonanilide anti-arrhythmic drug.
Methods and Results We individually substituted 24 residues in the pore helix and S6 domain of HERG to alanine. The currents through the mutant HERG channels expressed in Xenopus oocytes were measured with the standard two-microelectrode voltage clamp technique. The effect of ~85% inhibitory concentration (11 μM) of nifekalant was assessed with each mutant. Three mutations located in the pore helix (T623A, S624A, and V625A) and 3 mutations in the S6 domain (G648A, Y652A, and F656A) decreased the channel's sensitivity to nifekalant.
Conclusions The six identified residues likely form nifekalant-binding sites in a HERG channel as is the case for MK-499. [Jpn J Physiol 54 Suppl:S126 (2004)]

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HL-1 cells are cardiac myocytes derived from the atria of a transgenic mouse expressing the simian virus 40 (SV40) large T antigen and retains the adult differentiated cardiomyocyte phenotype. The present study was designed to characterize the K⁺ channel current in HL-1 cells using the whole-cell patch-clamp methods. In the presence of 0.4 μM nisoldipine, depolarizing voltage steps applied from a holding potential of –50 mV elicited the time-dependent outward current, followed by the decaying outward tail current upon return to the holding potential. The amplitude of the outward current was increased with depolarizations up to 0 mV but was then progressively decreased with further depolarizations. E-4031 (5 μM) or dofetilide (1 μM) almost totally abolished the time-dependent outward current as well as the tail current, suggesting that the outward current activated by depolarizations in HL-1 cells was mostly attributable to the rapidly activating delayed rectifier K⁺ current (I_Kr). The inhibitory effect of E-4031 and dofetilide was concentration-dependent with IC₅₀ of 21.1 and 15.1 nM, respectively. I_Kr in HL-1 cells was activated in response to membrane depolarization with V_1/2 of –20.4 mV. I_Kr in HL-1 cells also exhibited voltage-dependent inactivation during membrane depolarization with V_1/2 of –47.5 mV. Thus, properties of I_Kr in HL-1 cells are comparable to those of I_Kr in native mammalian cardiac cells and HL-1 cells provide a suitable experimental model for studies of native cardiac I_Kr. [Jpn J Physiol 54 Suppl:S127 (2004)]

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L-type Ca²⁺ channels play a crucial role in the contraction and the spontaneous activity of the heart. It has been reported that in sinoatrial node cells the L-type Ca²⁺ channels are derived from not only α1C(Cav1.2) but also α1D (Cav1.3). In this study we have investigated the molecular and electrophysiological properties of L-type Ca²⁺ channels in porcine sinoatrial node cells, in comparison to those of ventricular cells. In the patch clamp analysis, the L-type Ca²⁺ current in sinoatrial node cells was activated at more negative potential range than ventricular cells, peaking at 0 mV. Furthermore, the L-type Ca²⁺ current showed a retarded inactivation with a sustained component between -30 and +30 mV in sinoatrial node cells. Western blot analysis revealed the existence of α1D in sinoatrial node but failed to detect it in ventricles. On the other hand, α1C, which was predominant in ventricles, was only detectable in sinoatrial node. Dihydropyridine binding was examined and showed a K_D value of approximately 0.2 nM in both sinoatrial node and ventricles. The results indicate that a1D plays a major role as the L-type Ca²⁺ current in sinoatrial node cells, and may account for the different kinetics of L-type Ca²⁺ current between sinoatrial node and ventricular cells. [Jpn J Physiol 54 Suppl:S127 (2004)]

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Ca²⁺-dependent facilitation and inactivation are displayed in L-type Ca²⁺ channel in response to increased intracellular [Ca²⁺]i. CaM has been suggested to mediate the two autoregulatory feedbacks. We investigated this hypothesis using patch-clamp method (cell-attached mode) in guinea-pig ventricular myocytes. Ca²⁺ channnel activity was potentiated (facilitation) and then suppressed (inactivation) during continuous superfusion of high-Ca²⁺ solution. Both facilitation and inactivation were inhibited by CaM inhibitor, chlorpromazine (1, 10, 100μM) in a dose-dependent manner, implying that CaM is necessary for the two autoregulatory feedback mechanisms. On the other hand, CaMKII only affected the time course of facilitation and inactivation probably by slowing Ca²⁺ increase. These results suggest that CaM plays an important role in both facilitation and inactivation of cardiac L-type Ca²⁺ channel. [Jpn J Physiol 54 Suppl:S127 (2004)]

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In cardiac myocytes Ca²⁺ entry into the cells through Ca²⁺ channels play a crucial role in excitation-contraction coupling. PKA is the most important regulatory factor of the Ca²⁺ channel. Although PKA enhances Ca²⁺ channel activity in whole-cell condition, such regulation is not seen in excised patches, suggesting that the PKA effect requires cooperation with other factors. To confirm this hypothesis, we have investigated a possible involvement of CaM in the PKA effect in guinea-pig cardiac ventricular myocytes. Our results showed CaM inhibitor chlorpromazine (CPZ) abolished the effect of 8-Br-cAMP on the channel in the cell-attached mode. In inside-out patches, PKA catalytic subunit (PKAc)+ATP had no effect on the channel activity, but when channel activity was maintained by CaM+ATP, subsequent addition of PKA significantly increased the channel activity. Meanwhile, phosphorylation with PKA prevents the time-dependent attenuation of the CaM effect. These results indicate that PKA modulation of Ca²⁺ channel requires coordination with CaM. [Jpn J Physiol 54 Suppl:S127 (2004)]

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It was reported that gap junction channels exist between primary sensory neurons in the mesencephalic trigeminal nucleus (MesV) that innervate jaw-closing muscle spindles and periodontal mechanoreceptors. Recently, the gating kinetics of gap junction channels has been well investigated in insect cell lines and Xenopus oocytes. Here we focused on the electrophysiological properties of gap junction channels between MesV neurons. We made dual whole-cell patch-clamp recordings from such a pair of MesV neurons that contacted each other with a large area of plasma membrane, using slice preparations of 10-17 day old rats. We applied ramp pulses (0.1-4 V/sec) to one cell under V-clamp conditions and recorded the responses in the other cell under V-clamp and I-clamp conditions. Of 9 paired recordings, 4 pairs showed inward rectification with low coupling ratio, while 5 pairs showed outward rectification with high coupling ratio. There was significant delay between the onset of voltage pulses in stimulating cells and the peak responses in recording cells, revealing that the gating process of gap junction channels is slow. Especially, in the outwardly rectified coupling, the activation of outward gap junction current (e.g. 6.0±2.4 msec at 0.5 V/sec ramp pulse) is slower than the deactivation (e.g. 3.1±1.8 msec at 0.5V/sec ramp pulse). The maximum coupling conductance of these channels is approximately 5.5±1.1 nS, suggesting that 10-20 gap junction channels contribute to the gap junction current between the pair of coupled MesV neurons. [Jpn J Physiol 54 Suppl:S128 (2004)]

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Effects of intracellular Mg²⁺ ([Mg²⁺]_i) on gating kinetics of the native Ca²⁺-activated and voltage-sensitive BK channel in cultured human proximal tubule cells (RPTECs) were analyzed using the inside-out mode of the patch-clamp technique. At constant intracellular Ca²⁺ ([Ca²⁺]_i, 10⁻⁵ - 10⁻⁴ M), open probability (P_o) of the channel was raised by adding [Mg²⁺]_i (1 - 10 mM), which shifted the P_o - V_m relationship to the negative voltage direction without change in the value of gating charge. At constant V_m, increasing [Mg²⁺]_i to 10 mM raised P_o and enhanced sigmoidicity of the P_o - [Ca²⁺]_i relationship with elevation of the Hill coefficient. These would suggest that the [Mg²⁺]_i-induced changes in P_o resulted from changes in [Ca²⁺]_i-sensitivity. However, increasing [Mg²⁺]_i from 0 to 10 mM at 10⁻⁵ M [Ca²⁺]_i raised P_o by extension of the open time, whereas increasing [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M without [Mg²⁺]_i raised P_o mainly by shortening the closed time, even though these two maneuvers induced similar elevation of P_o. Moreover, adding 10 mM [Mg²⁺]_i at a low [Ca²⁺]_i (10^−5.5 M) extended the closed time as well as the open time. We conclude that the major action of raising [Mg²⁺]_i on the BK channel in RPTECs is to extend the open time, which would be distinct from the changes in P_o by voltage or [Ca²⁺]_i. The extended closed time at the low [Ca²⁺]_i might result from reduction of the Ca²⁺-sensitivity probably due to a competitive inhibition of Mg²⁺ to high affinity Ca²⁺-binding sites. [Jpn J Physiol 54 Suppl:S128 (2004)]

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The Ca²⁺-activated BK channel in cultured human proximal tubule cells (RPTECs) is sensitive to voltage, ATP, and pH_i. Furthermore, we recently found out that this channel was regulated also by intracellular Mg²⁺ ([Mg²⁺]_i). One of the significant effects of [Mg²⁺]_i on channel was to extend the open time at a low intracellular Ca²⁺ ([Ca²⁺]_i). However, [Mg²⁺]_i effect on channel under the relatively high [Ca²⁺]_i is still unknown. In this study, we examined effects of [Mg²⁺]_i (1 - 10 mM) on open probability (P_o) and gating mechanisms of the BK channel in the presence of 0.1 - 1.0 mM [Ca²⁺]_i, and compared with the gating of high [Ca²⁺]_i-induced channel activation, using the patch-clamp technique. When the channel was activated by 0.1 mM [Ca²⁺]_i, raising [Mg²⁺]_i up to 10 mM elevated P_o and induced parallel shifts of P_o - V_m relationships. These effects of [Mg²⁺]_i on channel P_o and the P_o - V_m relationships were mimicked by either total or partial substitution of equimolar [Mg²⁺]_i to [Ca²⁺]_i. In the presence of 1 mM [Ca²⁺]_i, addition of 10 mM [Mg²⁺]_i further enhanced channel P_o, which was also similar to channel activation by raising [Ca²⁺]_i to 10 mM. The dwell-time histograms obtained from 10 mM [Mg²⁺]_i in the presence of 0.1 mM [Ca²⁺]_iwere similar to that obtained from 10 mM [Ca²⁺]_i. These results suggest that, in addition to high affinity Ca²⁺-binding sites, the BK channel in RPTECs possesses the binding site for both Ca²⁺ and Mg²⁺, which is involved in channel activation, and its affinity ratio of Ca²⁺/ Mg²⁺ is close to 1. [Jpn J Physiol 54 Suppl:S128 (2004)]

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Mechanosensitive channels (MSCs) have been suggested to play crucial roles in cell physiology, however, never clarified yet. One of the reasons is the lack of specific blockers to MSCs. To date gadolinium and aminoglycosides have been used as potent but nonspecific blockers to the MSCs. Very recently, the peptide GsMTx-4 from spider (Grammostola spatulata) venom was reported to be a promising candidate for a specific blocker of MSCs. However, its actions on MSCs are poorly characterized. Here we report that GsMTx-4 can block the stretch activated and Ca²⁺-activated big K channel (SAKcaC) from chick embryonic myocardial cells. Single channel currents of the SAKcaC were recorded mostly from excised inside-out patches and negative pressure was applied in the pipette to stretch the patch. Open probability of the channel (Po) was significantly reduced by the μM range of GsTMx-4 without changing its single channel conductance, suggesting that the peptide blocks the channel in a slow blockade manner. Whether GsTMx-4 modifies the mechano-gating of SAKcaC or not is under investigation. GsTMx-4 also blocked the SAKcaC, which was cloned and expressed in CHO cells, in a similar manner. However, GsTMx-4 slightly inhibited the mutated SAKcaC that lacks mechanosensitivity but preserved original voltage and Ca²⁺dependencies of the SAKcaC. Although we do not know this mechanism yet, the last result provides a possibility that GsTMx-4 would work as a blocker specific to MSCs. [Jpn J Physiol 54 Suppl:S128 (2004)]

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MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and has been proposed to play major role in the protection against rapid osmotic downshock. Although the gene has been cloned and X-ray crystallographic analysis has identified the 3D structure of the open channel, much less is known how MscS senses membrane tension. In this study, we examined the function of the mutants generated by scanning mutagenesis to identify the amino acid residues responsible for sensing membrane tension. Channel functions were examined by the patch clamp (in vitro) and hypo-osmotic shock (in vivo) methods. A hydrophilic amino acid (asparagine) replaced one of the hydrophobic residues that are thought to make contact with the lipid bilayer. Replacement of the hydrophobic residues near the periplasmic or cytoplasmic end of TM1 or TM2 transmembrane domain decreased the sensitivity of MscS to tension in the inside-out patch membrane from giant spheroplasts of E.coli. Substitution of the residues located around the center of lipid bilayer did not impair the channel's mechanosensitivity. The survival rate of bacteria challenged to hypo-osmotic shock showed comparative results with those of in vitro experiments. These findings suggest that the interaction between lipid and the both ends of TM1 and TM2 is important for tension sensing. [Jpn J Physiol 54 Suppl:S129 (2004)]

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Background: Computer searches for human genome revealed a family of novel G-protein coupled receptors, THTRs, which are highly homologous to a family of bitter taste receptors, T2Rs (Takeda et al. (2002) FEBS Lett. 520, 97). We have previously cloned all human THTR and T2R genes. We have also shown that the assay system using receptor-Gα16 fusion proteins is an effective method for ligand screening. In this study, this assay system was utilized to identify ligands of THTRs and T2Rs. Method: THTR- and T2R-Gα16 fusion protein cDNA were constructed and stably expressed in CHO cells. The cells were stimulated with various stimulants, including tastants. Subsequently, The supernatant of the cells was collected and prostaglandin E₂ generation was measured using an enzyme immunoassay kit. Tissue distribution of receptors was investigated by RT-PCR. Results & Conclusion: THTR 5 was highly expressed in tongue epithelia. In cells, expressing THTR5-Gα16 fusion protein, glucose induced marked prostaglandin E₂ generation. These data suggested that THTR5 might function as glucose receptor in tongue epithelia. [Jpn J Physiol 54 Suppl:S129 (2004)]

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The regulators of G protein signaling (RGS) proteins are a large family of evolutionarily conserved proteins that function as negative regulators of GTP-binding proteins, and we previously reported that they also function as kinetic accelerators of turning -on and -off of G protein mediated responses. We isolated RGS related cDNA clones from medaka fish skin with a view to seeking RGSs related to actual physiological phenomena, i.e. changes of the skin color. MeRGS2, homologous to mammalian RGS2, was primarily localized in the heart and the skin of medaka fish. MeRGS2 exhibited uniform subcellular distribution under basal conditions, and it translocated to cell membrane when constitutively active mutant of Gαo was co-expressed. MeRGS2 was co-immunoprecipitated with an activated Gαq and Gαi/o protein. We next analyzed electrophysiologically the effects of MeRGS2 on Gq- and Gi/o- mediated responses under the two-electrode voltage clamp in Xenopus oocytes. When MeRGS2 was co-expressed with Gq-coupled muscarinic acetylcholine (mACh) type 1 receptor, it strongly suppressed Gq-mediated Ca²⁺ activated Cl⁻ current in an expression level dependent manner. By co-expressing MeRGS2 with G protein-gated inwardly rectifying K⁺ (GIRK) 1/2 channels and Gi/o-coupled mACh type 2 receptor, it accelerated the turning -on and -off of Gi/o-mediated modulation of GIRK current. These biochemical and electrophysiological results show that MeRGS2 serves as RGS for both Gq and Gi/o proteins. [Jpn J Physiol 54 Suppl:S129 (2004)]

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The K-ATP channel plays a mojor role in coupling the metabolic state to the electrical activity of plasma membranes in several cells. Endosulfine was identified as the endogenious regulator of K-ATP channels. Endosulfine belongs to a highly conserved family of aAMP-regulated phosphoprotein (ARPP) that includes ARPP-16, ARPP-19, ARPP-21 and DARPP-32. The biological function of endosulfine/ARPP has not been understood. In this study, we examined the mechanisms of effects of endosulfine/ARPP on K-ATP channels. Inside-out patchclamp analysis showed that exogenously addministered endosulfine inhibited the current of K-ATP channels of pancreatic beta-cells, while ARPP had no effect. The cell-attached patchclamp analysis showed that the beta-cell transfected with adenovirus expressing endosulfine exhibited action potentials at low glucose states. This result indicates that not only ARPP but also endosulfine reacts with the K-ATP channel as an intracellular ligand to inhibit its channel activity. However, endogenous endosulfine is expressed in pancreatic delta-cells but not beta-cells. Since the threshold glucose concentration for activation of delta-cells is lower than that of beta-cells, the higher sensitivity for glucose in delta-cells may be in part due to the expression of and regulation by endosulfine. [Jpn J Physiol 54 Suppl:S129 (2004)]

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The supraoptic nucleus is neurosecretory cell which secretes oxytocin and vasopressin in the neurohypophysis. Slice patch clamp teqnique was applied in order to measure the whole cell currents and membrane potentials using male rat in postnatal 21-28. The supraoptic nucleus was identified under infra-red microscopy. Under current clamp condition, ATP application depolarized and after application, the firing frequency was suppressed. ATP application (100 μM) activated the rapid inward currents and slowly desensitized.at the holding potential of -50 mV. This inward current was blocked by P2 antagonist, suramin and pyridoxal - phosphate - 6 - azophenyl - 2' ,4'- disulfonate (PPADS). ATP analogue, a,b methylene ATP (100 μM) also evoked inward current. The peak values of a,b methylen ATP-evoked response showed 10-20% of those of ATP-evoked ones. After patch-clamp recording, suction was applied to aspirate the contents of the cell into the pipette. Subsequently, single cell RT-PCR analysis for all P2X subtypes was performed using harvested cytoplasm. Signal of P2X2 subtype was detected in cells which responded to ATP. In supraoptic nucleus, P2X2 subtype was mainly expressed and functioned. [Jpn J Physiol 54 Suppl:S130 (2004)]

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Recently, we have discovered a novel phosphatase-like gene from ascidian and named as CiVSP (Ciona intestinalis voltage sensor-containing phoshatase). This protein consists of two domains; the putative voltage sensor domain and the phosphatase domain. The voltage sensor domain showed high similarity to the transmembrane region of voltage-gated channels. Indeed, the Xenopus oocyte injected with CiVSP cRNA generated gating current in response to membrane depolarization by two-electrode voltage clamp. The phosphatase domain showed high similarity to PTEN (phosphatase and tensin homolog deleted from chromosome ten), a phosphatase which dephosphorylate the phosphate at the 3'-position of phosphatidylinositol 3,4,5-trisphosphate (PIP₃). In order to examine whether CiVSP also dephosphorylates PIP₃ like PTEN, we performed in vitro phosphoinositide phosphatase assay by colorimetric method using malachite green. We found that the CiVSP possessed the phosphatase activity to PIP₃ like PTEN. The substitution of cystein residue at the position 363, which corresponds to the active site of PTEN, to serine resulted in a complete loss of phosphatase activity. In order to know the substrate specificity of CiVSP for various phosphoinositides, we expressed the phosphatase domain fused to glutathione S-transferase (GST) in E.coli , purified and performed in vitro phosphatase assay. Further, the phosphatase activity of CiVSP expressed in mammalian cells was also investigated. [Jpn J Physiol 54 Suppl:S130 (2004)]

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In our systematic survey of channel-like genes from the whole genome sequence of Ciona intestinalis, one of the ascidians, we found a gene with weak homology to a voltage-gated sodium channel. Its cDNA was cloned by RT-PCR from young adult. This protein contains an enzyme-like region instead of pore-forming region following the four transmembrane regions. The fourth transmembrane segment of this protein showed significant homology to the S4-segment of the voltage-gated channels, which is known to contain several positively charged amino acid residues periodically located at every third position and move across the membrane according to the membrane potential change. Therefore, we named this Ciona protein CiVSP (voltage-sensor containing protein). To test if the S4-like region works as voltage sensor in CiVSP, cRNA of CiVSP was injected to Xenopus oocyte and gating current was tried to be recorded using the two electrode voltage-clamp technique and the cut-open oocyte voltage-clamp technique. Robust gating current with voltage dependency was detected. The G-V curve of ON-currents showed similar voltage-dependency and the magnitude to those of OFF-currents. The truncated protein, lacking the C-terminal enzyme domain, still showed gating current. Further, when two of four positive charged amino acid residues in the S4-like segment were mutated to neutral ones, gating current was completely lost. These results suggest that voltage sensor of CiVSP operates in a similar way as that of voltage-gated channels. [Jpn J Physiol 54 Suppl:S130 (2004)]

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When we surveyed ion channel genes from the genome sequence of a tunicate, Ciona intestinalis, we discovered a novel putative membrane protein which has a voltage sensor motif with significant homology to voltage-gated channels, and a phosphatase domain just downstream of the transmembrane region. We termed it CiVSP (Ciona Voltage Sensor-containing Phosphatase). In fact, VSP showed phosphoinositide phosphatase activity and gating currents. We examined whether the voltage sensor functionally couples with the phosphatase domain of CiVSP. cRNAs encoding CiVSP and several types of phosphoinositide-sensitive ion channels were coinjected into Xenopus oocyte and the two-electrode voltage clamp recording was performed. Changes of the phosphatase activity with membrane potentials were detected by monitoring changes of ion currents through exogenously expressed channels. Channel activities were increased after hyperpolarization and were decreased by depolarization. It suggests that phosphatase activity of CiVSP is higher in a hyperpolarized condition than in a depolarized condition. These phenomena were never observed in oocytes injected only with channel cRNAs and in oocytes coinjected with a mutant CiVSP in which the phosphatase activity was eliminated. These results strongly suggest that CiVSP is a Voltage-Sensitive Phosphatase. CiVSP is the first report of a membrane protein which can transduce electrical signal into chemical signal without requiring ionic flow through membrane. [Jpn J Physiol 54 Suppl:S130 (2004)]

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We examined effects of prostaglandin E₂ (PGE₂) on electrical and morphological properties of rat osteoclasts. PGE₂ (>10 nM) stimulated an outwardly rectifying Cl⁻ current and caused a long-lasting depolarization of cell membrane. This PGE₂-induced Cl⁻ current was reversibly inhibited by DIDS, NPPB and tamoxifen. The anion permeability sequence of this current was I⁻>Br⁻>=Cl⁻>gluconate⁻. When the Cl⁻ current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE₂ was observed. Forskolin and dibutyryl-cAMP mimicked the effect of PGE₂. The PGE₂-induced Cl⁻ current was inhibited by pretreatment with GDPβS, Rp-cAMPS and H89. PGE₂ (1 μM) also reduced cell surface area and suppressed motility of osteoclasts, and these effects were inhibited by Rp-cAMPS or H89. PGE₂ is known to exert its effects through four subtypes of PGE receptors (EP1, EP2, EP3 and EP4). The EP2 (ONO-AE1-259) and EP4 (ONO-AE1-329) agonists, but not EP1 (ONO-DI-004) and EP3 (ONO-AE-248) agonists mimicked the electrical and morphological effcts of PGE₂ on osteoclasts. These results show that PGE₂ stimulates rat osteoclast Cl⁻ current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption. [Jpn J Physiol 54 Suppl:S131 (2004)]

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We previously showed that cultured cortical neurons possess the volume-sensitive Cl⁻ channel which serves as a Cl⁻ efflux pathway for the regulatory volume decrease after hypotonic swelling. It has been reported that neurons show swelling coupled to Na⁺ influx via NMDA receptors under excitotoxic conditions. In this study we investigated whether an excitotoxic insult activates volume-sensitive Cl⁻ channels in mouse cortical neurons. Exposure to a glutamate receptor agonist, NMDA, activated whole-cell currents that exhibited anion selectivity, outward rectification, inactivation kinetics at large positive potentials and sensitivity to a Cl⁻ channel blocker, NPPB. NMDA-activated anion currents were suppressed by osmotic shrinkage induced by hypertonic solution or by removal of extracellular Na⁺. Morphological observations in calcein-loaded neurons showed that not only somatic swelling but also varicosity formation on the dendrites were induced by exposure to NMDA. After washout of NMDA, varicosities were found to gradually disappear. Such recovery from dendritic beading was not observed in the presence of NPPB. Taken together, it is suggested that the volume-sensitive outwardly rectifying Cl⁻ channel plays a role in recovery from excitotoxic varicosity formation in cortical neurons. [Jpn J Physiol 54 Suppl:S131 (2004)]

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Human epithelial Intestine 407 cells respond to hypotonic stimulation with activation of the volume-sensitive outwardly rectifying (VSOR) chloride channels. In cell-attached patches, the VSOR single-channel currents exhibit outward rectification and voltage-dependent inactivation at large positive potentials. We attempted to estimate the dimensions of the VSOR channel pore using a nonelectrolyte partitioning method. Effects of nonelectrolytes with different molecular sizes on the cell-attached single-channel events were observed by applying from the extracellular side. Ethylene glycol (R_h=2.7 Å), triethylene glycol (R_h=2.7 Å) and polyethylene glycols of molecular weight 200-300 (PEG 200-300: R_h=4.5-5.3 Å) effectively suppressed the single-channel outward current, whereas larger molecules (PEG 400-4000 with R_h=6.2-19.1 Å) had little or no effect on the channel current amplitude. Since all the molecules tested effectively decreased electro-conductivity of the bulk solution, it appears that the observed differential effect between PEG 200-300 and PEG 400-4000 on the VSOR single-channel current was due to their limited partitioning into the channel lumen. From the cut-off radius of the PEG molecules, the VSOR channel pore is assessed to be 6.3 Å. [Jpn J Physiol 54 Suppl:S131 (2004)]

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A sheet of endothelial cells was isolated from a guinea-pig mesenteric artery and placed on a cover slip with the abluminal surface up. Two patch electrodes were applied on two adjacent endothelial cells which should be coupled electrically with gap junctions. The patch membrane was ruptured in one electrode and the membrane potential was monitored in the current clamp mode. The other electrode was used in the cell-attached patch-clamp mode and the patch membrane potential was controlled using the membrane potential recorded through the first electrode. The membrane potential was usually less negative than -10 mV and the cells seemed to have lost the intracellular K⁺. Incubation in a 150 mM-K⁺ solution for 10-15 min was employed to recover the high intracellular K⁺ concentration and acetylcholine (ACh, 3 μM) was applied after the normal extracellular solution was reintroduced. ACh induced a transient hyperpolarization and openings of 2 pS channels. The channel had a reversal potential of -70 mV and did not open in the presence of apamin (100 nM). This channel was distinct from the nonselective cation channels which were also activated by ACh, and seemed to be the K⁺ channel responsible to the ACh-induced hyperpolarization. [Jpn J Physiol 54 Suppl:S131 (2004)]

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The subfornical organ (SFO) is a forebrain structure that is related to drinking behavior, cardiovascular regulation, vasopressin release and sodium appetite. It has been recently reported that SFO neurons projecting to hypothalamus have large I_A. We electrophysiologically classified acute dissociated SFO neurons (n=60) under the presence of 300 nM tetrodotoxin (TTX) into three types; Type-I, -II and -III neurons showed fast exponential decay components (time constant, τ<100 ms, n=9), slower (100 ms<τ<400 ms, n=17) and further slower (τ>500 ms, n=34) at +20 mV voltage-step from -70 mV, respectively. As some Type-III neurons (n=10) showed fast exponential decay components by the addition of CdCl₂, they were further classified to Type-IIIa and other neurons were classified to Type-IIIb. Under the presence of TTX and CdCl₂, I_As were isolated by -40 mV prepulse before voltage-steps. Some Type-IIIb neurons (n=7) did not show I_A. Although all Type-I and -IIIa neurons showed larger I_A; rates of the peak amplitudes of I_A vs that of whole-cell currents were over 0.43. RT-PCR assay revealed that the SFO has mRNA of Kv1.4, Kvβ1, Kv3.4, Kv4.1, Kv4.2, Kv4.3, but not Kv3.3. All Type-II neurons showed exponential slowly decay components that was not inhibited by -40 mV prepulse and 1 mM tetraethylammonium (TEA) sensitive (presumed Kv3.4). Under the presence of TTX, CdCl₂ and TEA, recovery rate of I_A fitted to double-exponential function; τ of rapid component was under 300 ms (presumed Kv4-family) and τ of slow component was over 1 s (presumed Kv1-family). [Jpn J Physiol 54 Suppl:S132 (2004)]

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